Heart-cutting steroids: better, faster, stronger

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  • Published: Dec 13, 2016
  • Author: Ryan De Vooght-Johnson
  • Suppliers: Thermo Fisher Scientific
  • Channels: Gas Chromatography
thumbnail image: Heart-cutting steroids: better, faster, stronger

Roid rage

Performance-enhancing drug abuse is prominent in sport. Rigorous measures already exist to dissuade athletes from exploiting drugs for performance benefits…but can we keep steroid use at bay? Image by Psychonaught. Reproduced from Wikipedia under Creative Commons license.

From Floyd Landis’ sky-scraping testosterone levels in the 2006 Tour de France to the recent scandal regarding doped up Russian Olympians, performance-enhancing drug abuse is prominent in sport. Rigorous measures already exist to dissuade athletes from exploiting drugs for performance benefits…but can we keep steroid use at bay?

The World Anti-Doping Agency (WADA) finds itself in perpetual conflict with teams and associations over the use of anabolic steroids. Trace-level effectiveness and swift in vivo catabolism make these substances very difficult to manage. With the natural presence of steroids in humans and considerable inter-individual variation, the conundrum is further exacerbated.

Fortunately, differences between exogenous and endogenous steroids can be distinguished by GC/MS applied to urine samples; the carbon isotope ratio (CIR) of synthetic samples does not match that of natural forms.

Highly sensitive, high-tech screening techniques by GC-MS/MS allow for ‘suspicious sample’ identification. Those picked up on are subject to further GC/C/IRMS analysis to confirm the presence of a banned substance and to verify their levels.

Heart-cut clarity

The main restriction of GC/C/IRMS involves the time and regular monitoring required through the sample preparation phase. Identification of this limitation led a combined force of Germans and Brazilians to optimise current methods. Consequently, they devised an efficient, selective technique of automatic sample analysis, allowing for more rapid turnover.

The group, directed by Alessandro Casilli of the Brazilian Laboratory Doping Control, replaced two phases of conventionally applied HPLC with multi-dimensional GC during sample preparation. MDGC, a technique developed for the separation of more complex matrices, involves the connection of two GC columns in series. By ensuring column orthogonality, complete matrix separation can be obtained. This technique also increases maximum peak capacity, intensifying chromatographic resolution.

The team prepared urine aliquots of the steroids to be examined: androsterone, etiocholnolone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol and testosterone. In a paper published in Drug Testing and Analysis, Casilli explains how “all samples were analysed on a Thermo Multidimensional Gas Chromatograph coupled to a DeltaV gas isotope ratio mass spectrometer via a GC combustion interface and an ISQ quadrupole mass spectrometer.”

The 1D column (TR-1 MS, 100% dimethylpolysiloxane), monitored by a flame ionisation detector, was heated from 100 °C to 300 °C at a rate of 25 °C/min. Peaks of interest were ‘heart-cut’ from this non-polar, stationary phase to the 2D column (HP-17, 50% phenyl–50% dimethylpolysiloxane). The second dimension, monitored by quadrupole MS and IRMS, was run with the GC oven set to 100 °C for almost an hour. A 25 °C/min ramp was applied until 250 °C, followed by a 3 °C/min increase up to 300 °C. A Thermo IQ was coupled to the MDGC/C/IRMS for comparison of MS data with pure steroid standards.

This heart-cutting approach improves peak separation by reducing interference usually caused by nearby peaks. The complex nature of urine as a matrix made this vital to the team; hundreds of peaks were observed over a run time of just 90 minutes.

Reaching a new high

Casilli and his team obtained promising results; CIR data proved comparable to that obtained by routine HPLC methods. The authors have provided a system enabling “simultaneous communication between all parts offering the possibility to work 24 hours for 7 days a week, without any manual intervention besides the preparation of the samples and the sequence of analysis.” These advances enable the more rapid processing of higher volumes of urine samples for IRMS so the throughput of steroid level analysis from human urine samples could reach a new high.

Standards enforced by the World Anti-Doping Agency require further method validation from the researchers. Having taken this on board, Casilli’s group intend to ensure the “long term robustness” of their tests and broaden the scope of examinable steroids.

Related Links

Drug Testing and Analysis, 2016, 8, Early View paper. Casilli et al. Optimization of an online heart-cutting multidimensional gas chromatography clean-up step for isotopic ratio mass spectrometry and simultaneous quadrupole mass spectrometry measurements of endogenous anabolic steroid in urine.

Chromatography Online, A Short Introduction to MDGC

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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